Itch, one the most distressing of dermatological symptoms, is associated with numerous skin diseases, such as atopic dermatitis, as well as with extracutaneous disorders. Intense scratching causes skin damage, which further increases itchiness. Unfortunately, currently available itch medications are ineffective in many chronic itch conditions and often cause undesirable side effects. Despite its clinical importance, the etiology and neurobiology of itch are not well understood. The current application is designed to characterize neuronal mechanisms for itch in both the epidermis and the brain. Specific Aim 1 will focus on elucidating the function of two subpopulations of cutaneous afferent neurons that are marked by the expression of either a G protein- coupled receptor, Mrgprd, or an ion channel, TRPV1. These two subpopulations are distinct in their expression of sensory molecules, central and peripheral projection patterns within the epidermis and the spinal cord, and roles in nociception. The two subpopulations will be ablated or silenced, individually or together, in adult mice by using genetic and pharmacological methods, and the effect of these manipulations on scratching behavior toward pruritogen-exposed skin will be assessed. This experiment will test whether Mrgprd+ and TRPV1+ neurons are required for acute itch. Furthermore, the role of these neurons in chronic itch will be analyzed using mouse models that mimic atopic dermatitis of human patients, by scoring spontaneous as well as pruritogen-induced scratching. Specific Aim 2 will systemically explore the brain regions that are responsible for pruritogen-induced scratching behavior. The candidate brain regions will be identified using the neuronal activity marker c-fos, and then functionally characterized by acutely silencing or activating the brain regions, using neuronal circuit manipulation techniques such as the ivermectin-gated chloride channel, GluCl12, and the light-gated ion channel, channelrhodopsin-2. In parallel, itch-mediating regions in the brain will be identified using anterograde viral tracing from the itch-specific neuronal population in the spinal cord, marked by the expression of gastrin-releasing peptide receptor. Successful completion of the proposed studies will substantially increase our knowledge of how pruritogens are detected by primary afferent neurons and how itch signals from the peripheral afferent neurons are processed in the brain to generate scratching behavior. During the first 2 years, the Principal Investigator (PI) will continue to work under the guidance of Dr. David Anderson at the California Institute of Technology to perform the proposed studies in Specific Aim 1 and 2. At the same time, the PI will learn the professional skills and experimental techniques needed for the transition to independent investigator status and successful completion of Specific Aims 1 and 2 during a 3- year independent investigator period.